A causative role of high-risk human papillomavirus (HPV) types in human cancer has been established, particularly for those of the anogenital region including cervical and anal cancers. The etiologic course of such cancers is thought to involve HPV infection, persistence of infection, progression to an immediate precursor of cancer, and finally invasion (the latter two steps requiring additional host oncogenic events). Despite the success of cervical cancer screening programs based on the appearance of abnormal cells in cytology specimens and HPV-type, there is a great need to identify additional biomarkers to increase the sensitivity with which precancer/cancer are detected, so providing additional triage of the approximately 2,000,000 women each year in the US who undergo colposcopy for follow-up of abnormal cytology. Gain of 3q and to a lesser extent gain of 5p and 20q, are genomic abnormalities commonly detected in HPV-associated cancers, and as such represent potential biomarkers of HPV-associated cancer progression. In the current application, Cancer Genetics, Inc. proposes to develop and validate a fluorescence in situ hybridization (FISH)-based HPV- associated cancer detection test (FHACT) to detect the three genomic abnormalities in cervical and anal cytology specimens. Phase I;Specific Aim 1: Confirmation of gain of 3q, 5p, and 20q in cervical precancerous and cancer cytology specimens. 200 cervical cytology specimens will be submitted to four- color FISH using probes to detect gain of 3q (red), 5p (green), and 20q (gold), to confirm association with cytologic severity and to provide an initial estimate of the sensitivity for precancer/cancer detection. If the sensitivity is comparable to that for cytology alone, then Phase II will be initiated. Phase II;Specific Aim 1: Validation of automated scanning for the FHACT in cervical cytology specimens. The scanning/scoring procedure for the FHACT will be automated in order to adapt the assay for increased output. Phase II;Specific Aim 2: Optimization of the FHACT in 1,000 cervical cytology specimens. The sensitivity and specificity of the FHACT will be determined for the entire dataset of 1,000 cervical cytology specimens and compared with that by cytology alone, after optimization of cut-offs, probe combinations, and numbers of nuclei to score. Phase II;Specific Aim 3: Optimization of the FHACT in 1,000 anal cytology specimens. Comparable studies to those above will be performed for anal cytology specimens, where standard-of-care screening programs have as yet to be developed for at-risk populations. Specimens will be obtained from Drs. Castle and Wentzensen, at the National Cancer Institute who are leading studies aimed at improving cancer prevention screening programs and identifying biomarkers of HPV-associated cancers. The overall goal of the project then is to develop and validate a robust, sensitive, and specific FISH-based test that together with standard cytology and HPV-typing will significantly contribute to more accurate detection of precancer/cancer, impacting standard-of-care recommendations in HPV-associated cancer screening programs. PUBLIC HEALTH RELEVANCE: Despite the success of cervical cancer prevention screening programs, there are many women in the US, who annually undergo medical procedures based on an abnormal Pap test and oncogenic virus test, which are later found to be unnecessary and costly, due to the lack of the sensitivity of these tests. Thus, in the current proposal a genetic-based precancer and cancer detection test is to be developed and validated, that uses residual Pap or anal test material. Implementation of this test into cervical and anal cancer prevention screening programs should provide an additional triage to identify those patients who require medical follow-up versus who do not, thereby reducing the performance of unnecessary and costly medical procedures, ultimately impacting population coverage of such programs in low resource countries.